Acetylcholinesterase (AChE) has long been an important marker for studying the structure and function of the neuromuscular junction. Studies of AChE have provided important new insights into the development, organization and regulation of this model synapse. We have recently discovered that AChE catalytic subunits can be rescued from degradation by non-catalytic subunits providing new insight into the regulation of assembly of this important synaptic component. We have also made novel observations on the translocation of acetylcholine receptors to cell surface AChE clusters. Our specific aims are now to 1) study the stabilization of newly synthesized AChE by the collagen-like tail and other proteins to understand how this contributes to regulation. This includes the interactions of ColQ with the muscle-specific kinase MuSK using co-expression and overexpression of these various proteins. 2) We will study the regulation of assembly of the synaptic ColQ AChE form consisting of three tetramers attached to the collagenic tail and the role of transcriptional and post-transcriptional mechanisms. Our overall hypothesis is that ColQ expression is rate limiting and trans-synaptic regulation acts primarily to regulate ColQ. These studies include retroviral vector-mediated gene delivery into myoblasts and the expression of shRNAs to alter the availability of ColQ subunits. We will also determine the role of membrane depolarization and second messengers in regulating ColQ expression at the transcriptional level. Our last aim 3) is to study the formation of the AChE-containing clusters on the surface of myotubes and the agrin-induced translocation of AChR to those clusters. Here we will co-express the major components of the AChE complex including dystroglycan, perlecan, rapsyn, ColQ and AChE to define the necessary components for trafficking ColQ AChE to the cell surface and assembling AChE clusters. We will then study the role of MuSK in assembling the AChE clusters and the reciprocal interactions between ColQ AChE and MuSK in clusters of synaptic basal lamina components. These studies will employ retroviral-mediated gene transfer and expression under control of the Tet promoter, as well as shRNAs to alter expression of specific synaptic components in the myotubes. These studies will provide novel information regarding the assembly of AChE in particular, and the neuromuscular synapse in general.